Beverage compositions containing Bacillus coagulans MTCC 5856

ABSTRACT

The present invention discloses beverage compositions comprising probiotic  Bacillus coagulans  and water soluble prebiotic fibers that have been subjected to treatments under extreme stress, temperature and pressure conditions like brewing or aeration wherein the spore viability is maintained post said treatments.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This is a non-provisional patent application claiming priority from U.S.Provisional Patent Application No. 62/511,412 filed on 26 May 2017.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention in general relates to probiotics. Morespecifically, the present invention relates to beverage compositionscomprising probiotic Bacillus coagulans strain MTCC 5856.

Description of Prior Art

Probiotics are gaining importance as a dietary supplement owing to theirability to modify the gut microflora yielding increased health benefits.These probiotics can survive the acidic environments of the stomach andsmall intestine, but are not observed to colonize the gastro-intestinaltract. Thus, there exists a need for constantly supplementing probioticsfor the therapeutic benefits to persist.

Beverages are widely consumed food constituents and can be looked uponas a source for co-administering probiotics. It must be noted thatbeverage compositions are subjected to treatments under extreme stress,temperature and pressure conditions like brewing or aeration wherein theviability of the probiotic spores should be maintained post treatment toresult in a successful administration.

United States Patent Application 20090232941 discloses a beveragecomposition comprising Bacillus coagulans strains GBI-30, GBI-20 andGBI-40. United States Patent Application 20130295226 discloses a fruitbeverage composition comprising Lactobacillus paracasei. Both theseapplications do not disclose the method of brewing and the survival ofthe probiotic bacteria in harsh conditions. Also, it is well known inthe scientific art that that biological effects of probiotics orproducts thereof are strain specific and cannot be generalised amonggenera, species and strains (Probiotics: In Depth/NCCIH, U.S. Departmentof Health and Human Services, National Institutes of Health). Hence,there still exists a need to find a probiotic strain that is moreefficient and viable when subjected to extreme stress, temperature andpressure conditions.

The present invention solves the above technical problem by disclosing aprobiotic strain that is viable and efficient when co-administered withbeverages.

The principle objective of the inventions is to disclose beveragecompositions comprising Bacillus coagulans MTCC 5856 wherein the sporeviability is maintained post treatments.

The present invention fulfils aforesaid objectives and provides furtherrelated advantages.

SUMMARY OF THE INVENTION

The present invention discloses beverage compositions that have beensubjected to treatments under extreme stress, temperature and pressureconditions like brewing or aeration and comprise Bacillus coagulans MTCC5856 spores and water soluble prebiotic fibers wherein the sporeviability is maintained post said treatments.

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

In a principle embodiment, the present invention discloses a beveragecomposition comprising Bacillus coagulans MTCC 5856 in the form of sporeand bacterium wherein said bacterial spore has the ability to surviveextreme stress, temperature and aeration treatments of said beveragecomposition. In a related embodiment, the beverage is selected from thegroup consisting of coffee, tea, fruit juice, aerated soda, malt, energydrinks, chocolate, cocoa, fruit drinks, smoothies, milk shakes, mineralwater, cola, cheese spread, honey, tomato ketchup, jam, mayonnaise,protein drink and ice cream. In another related embodiment, the sporesinclude viable or heat killed or dead spores of Bacillus coagulans MTCC5856. In yet another related embodiment, the bacterium includes viableor heat killed or dead or lysed vegetative cells of Bacillus coagulansMTCC 5856.

In another preferred embodiment, the invention discloses a coffeecomposition comprising Bacillus coagulans MTCC 5856 in the form of sporeand bacterium wherein said bacterial spore is heat resistant having morethan 95% viability after brewing coffee. In a related embodiment, thecoffee is selected from the group consisting of decaffeinated coffee,unroasted green coffee and roasted coffee. In another relatedembodiment, the spores include viable or heat killed or dead spores ofBacillus coagulans MTCC 5856. In yet another related embodiment, thebacterium includes viable or heat killed or dead or lysed vegetativecells of Bacillus coagulans MTCC 5856.

In another related embodiment, the invention discloses a tea compositioncomprising Bacillus coagulans MTCC 5856 in the form of spore andbacterium wherein the said bacterial spore is heat resistant having morethan 95% viability after brewing tea. In a related embodiment, the teaincludes green tea, black tea, oolong tea, yellow tea, White tea ordecaffeinated tea, herbal tea selected from the group consisting ofrosehip tea, chamomile tea, jiaogulan tea, peppermint tea, rooibos tea,ginger tea, ginseng tea, or lemon grass tea. In another relatedembodiment, the spores include viable or heat killed or dead spores ofBacillus coagulans MTCC 5856. In yet another related embodiment, thebacterium includes viable or heat killed or dead or lysed vegetativecells of Bacillus coagulans MTCC 5856.

In another embodiment, the fruit component of the fruit juice isselected from the group consisting of orange, apple, pears, strawberry,raspberry, cranberry, blue berry, apricot, pineapple, peach, banana,mango, lime, grape, tomato, pomegranate, papaya, tender coconut water,kiwi and a fruit mixture.

In another preferred embodiment, the invention discloses an aerated sodacomposition comprising Bacillus coagulans MTCC 5856 in the form of sporeand bacterium wherein the said bacterial spore has the ability tosurvive aeration. In another related embodiment, the spores includeviable or heat killed or dead spores of Bacillus coagulans MTCC 5856. Inyet another related embodiment, the bacterium includes viable or heatkilled or dead or lysed vegetative cells of Bacillus coagulans MTCC5856.

In another preferred embodiment, the present invention discloses asynergistic beverage composition comprising a water soluble prebioticfiber and Bacillus coagulans in the form of spore and bacterium whereinthe said bacterial spore has the ability to survive extreme stress,temperature and aeration treatments of said beverage composition. In arelated embodiment, the beverage is selected from the group consistingof coffee, tea, fruit juice, aerated soda, malt, energy drinks,chocolate, cocoa, fruit drinks, smoothies, milk shakes, mineral water,cola, cheese spread, honey, tomato ketchup, jam, mayonnaise, proteindrink and ice cream. In another related embodiment the water solubleprebiotic fiber is selected from the group consisting offructooligosaccharides (FOS), Galacto-oligosaccharide (GOS), Inulin andpolydextrose. In another related embodiment, the spores include viableor heat killed or dead spores of Bacillus coagulans. In yet anotherrelated embodiment, the bacterium includes viable or heat killed or deador lysed vegetative cells of Bacillus coagulans.

In another preferred embodiment, the invention discloses a synergisticcoffee composition comprising a prebiotic water soluble fiber andBacillus coagulans in the form of spore and bacterium wherein the saidbacterial spore is heat resistant having more than 95% viability afterbrewing coffee. In a related embodiment, coffee includes decaffeinatedcoffee, unroasted green coffee and roasted coffee. In another relatedembodiment the water soluble prebiotic fiber is selected from the groupconsisting of fructooligosaccharides (FOS), Galacto-oligosaccharide(GOS), Inulin and polydextrose. In another related embodiment, thespores include viable or heat killed or dead spores of Bacilluscoagulans. In yet another related embodiment, the bacterium includesviable or heat killed or dead or lysed vegetative cells of Bacilluscoagulans. In a preferable related embodiment, the Bacillus coagulansstrain is preferably Bacillus coagulans MTCC 5856 and also the strainsderived from Bacillus coagulans Hammer strain accession number ATCC31284 and ATCC 7050

In another related embodiment, the invention discloses a synergistic teacomposition comprising a prebiotic water soluble fiber and Bacilluscoagulans in the form of spore and bacterium wherein the said bacterialspore is heat resistant having more than 95% viability after brewingtea. In a related embodiment, the tea includes green tea, black tea,oolong tea, yellow tea, white tea or decaffeinated tea, herbal teaselected from the group consisting of rosehip tea, chamomile tea,jiaogulan tea, peppermint tea, rooibos tea, ginger tea, ginseng tea, orlemon grass tea. In another related embodiment the water solubleprobiotic fiber is selected from the group consisting offructooligosaccharides (FOS), Galacto-oligosaccharide (GOS), Inulin andpolydextrose. In another related embodiment, the spores include viableor heat killed or dead spores of Bacillus coagulans. In yet anotherrelated embodiment, the bacterium includes viable or heat killed or deador lysed vegetative cells of Bacillus coagulans. In a preferable relatedembodiment, the Bacillus coagulans strain is preferably Bacilluscoagulans MTCC 5856, and also the strains derived from Bacilluscoagulans Hammer strain accession number ATCC 31284 and ATCC 7050

The aforesaid most preferred embodiments incorporating the technicalfeatures and technical effects of instant invention, are explainedthrough illustrative examples herein under.

Example 1: Methods Employed for Brewing Coffee and Tea with Bacilluscoagulans MTCC 5856

Bacillus coagulans MTCC 5856 Stability During Coffee Brewing

Commercial preparation of Bacillus coagulans MTCC 5856 spores wasstandardized to 15×10⁹ cfu (spores) per gram and added to groundedunroasted green coffee or roasted coffee and blended at room temperaturewith low r.p.m. for 60 min. Further, 1.0 g of water soluble prebioticfibres (fructooligosaccharide, galacto-oligosaccharides, inulin andpolydextrose) were added to 4.0 gm of unroasted green coffee or roastedcoffee and blended at room temperature with low r.p.m. for 60 min (Table1). Approximately 5.0 g of the compositions were brewed using electricbrewer and tested for viable spore count as per method described earlier(Majeed et al., World J Microbiol Biotechnol (2016) 32; 60). Averagemean of spore viable counts are expressed in log₁₀ CFU.

TABLE 1 Formulation of Coffee beverages #Coffee and Total beverageexcipient + weight Bacillus coagulans MTCC Prebiotic Fibre* perFormulation 5856 (15 × 10⁹ spores/gm) (grams) serving 01 0.144 g 3.96 +0.04 + 0.0 5.144 02 0.072 g 3.96 + 0.04 + 1.0 5.072 03 0.144 g 3.96 +0.04 + 1.0 5.144 04 0.288 g 3.96 + 0.04 + 1.0 5.288 # where coffee isunroasted green coffee beans and Roasted coffee beans. *Where theprebiotic fibres are FOS, GOS, Inulin and polydextrose

Bacillus coagulans MTCC 5856 Stability During Tea (Black, Green andOolong Tea) Brewing

Commercial preparation of Bacillus coagulans MTCC 5856 was standardizedto 15×10⁹ cfu (spores) per gram and added to black, green or oolong teaand blended at room temperature with low r.p.m. for 60 min. Further, 1.0g of water soluble prebiotic fibres (fructooligosaccharide,galacto-oligosacharides, inulin and polydextrose) were added to 4.0 gmof black, green or oolong tea and blended at room temperature with lowr.p.m. for 60 min (Table 2). Approximately 5.0 g of these teacompositions were brewed using electric brewer and tested for viablespore count as per method described earlier (Majeed et al., World JMicrobiol Biotechnol (2016) 32:60). Average mean of spore viable countsare expressed in log 10 CFU.

TABLE 2 Formulation of tea beverages #Tea and beverage Total excipient +weight Bacillus coagulans MTCC Prebiotic Fibre* per Formulation 5856 (15× 10⁹ spores/gm) (grams) serving 01 0.15 g 3.96 + 0.04 + 0.0 4.15 020.075 g  3.96 + 0.04 + 1.0 5.075 03 0.15 g 3.96 + 0.04 + 1.0 5.15 040.30 g 3.96 + 0.04 + 1.0 5.30 # Where the tea are black, green andoolong. *Where the prebiotic fibres are FOS., GOS, Inulin andpolydextrose

Effect of Gastric Stress and Brewing Conditions on the Germination ofBacillus coagulans MTCC 5856 Spores

The effect of gastric stress on the Bacillus coagulans MTCC 5856 sporeswas studied by treating brewed coffee or tea (as described in aboveparagraph) in acidic conditions. The pH of brewed coffee or tea wasadjusted aseptically to 1.5 using 2 N HCl and incubated at 37° C. for 3h with low r.p.m. After 3 h of incubation, again pH was adjustedaseptically back to 7.0 using 2 N NaOH and 0.5% (w/v) ox bile was addedto tea or coffee flask. Oxygen reducing enzyme Oxyrase (Oxyrase® forBroth, Oxyrase, Inc. Mansfield, Ohio, USA) was also added to each flask.Further flasks were incubated at 37° C. for 24 h with low r.p.m. pHvalues at 0 h of incubation and after 24 h of incubation were alsorecorded. Optical density (OD) at 600 nm was recorded at 0 h and after24 of incubation by diluting the sample into sterilized demineralizedwater to 1:5.

Example 2: Results for Brewing Coffee and Tea with Bacillus coagulansMTCC 5856 Brewed Coffee with Bacillus coagulans MTCC 5856

The thermo-stability and compatibility of Bacillus coagulans MTCC 5856spores in coffee was evaluated during brewing using electric brewer.Stainless steel and paper filter were used in brewer to understand thefeasibility of probiotic brewing. There was 0.5 Log₁₀ cfu (spores)reduction after brewing which remained almost same even after 10 min ofincubation at room temperature (Table 3). The data suggested thatBacillus coagulans MTCC 5856 spores remained 94.83% viable after brewingusing Stainless steel filter. There was no significant differenceobserved when coffee was brewed using stainless steel or paper filters.

TABLE 3 Viability of B. coagulans MTCC 5856 during Coffee brewing usingelectric brewer Brewed and filtered Brewed and filtered using SS filterusing paper filter Log₁₀ Viability Viability Time CFU/10 g (%) Log₁₀CFU/10 g (%) Post Brew 9.8388 ± 0.012 100 9.8388 ± 0.012 100 After Brew9.3304 ± 0.011 94.83 9.2600 ± 0.014 94.11 After 10 min 9.3096 ± 0.01394.63 9.2504 ± 0.012 94.01

Brewed Tea with Bacillus coagulans MTCC 5856

Black tea, green tea and oolong tea were supplemented with Bacilluscoagulans MTCC 5856 spores and brewed to determine the spore survivalduring brewing. Bacillus coagulans MTCC 5856 spores were found to behighly stable and the viability remained above 99% after brewing (Table4 & 5).

TABLE 4 Viability of B. coagulans MTCC 5856 during black tea brewingusing electric brewer Formulation-01 Formulation-02 Formulation-03 Log₁₀CFU/Viability Log₁₀ CFU/Viability Log₁₀ CFU/Viability Time serving (%)serving (%) serving (%) Initial count 8.9309 ± 0.011 100.00 9.1182 ±0.012 100.00 9.4667 ± 0.010 100.00 Post Brew 9.1760 ± 0.014 102.749.1846 ± 0.011 100.73 9.6570 ± 0.014 102.01 After 10 min 9.1583 ± 0.011102.55 9.1760 ± 0.010 100.63 9.6683 ± 0.011 102.13 After 72 h chilling8.6570 ± 0.012  96.93 8.6739 ± 0.012  95.13 9.2878 ± 0.011  98.11

Same brewed tea samples were chilled for 72 h and then spore viabilitywas determined. The viability of spores in black, green and oolong teaafter chilling for 72 h was 95.13%, 93.58% and 94.51% respectively(Table 4 & 5).

TABLE 5 Viability of B. coagulans MTCC 5856 during green and oolong teabrewing using electric brewer Green Tea Oolong Tea Log₁₀ Viability Log₁₀Time CFU/serving (%) CFU/serving Viability (%) Post Brew 9.4471 ± 0.011100 9.4313 ± 0.012 100 After Brew 9.3830 ± 0.012 99.32 9.3921 ± 0.01199.58 After 10 min  9.371 ± 0.013 99.19 9.3842 ± 0.014 99.50 After 72 h 8.841 ± 0.014 93.58  8.914 ± 0.012 94.51 chilling

Germination of Spores

To deliver the right amount of viable Bacillus coagulans MTCC 5856spores in the human gut, the effect of brewing conditions and gastricstress on the germination of Bacillus coagulans MTCC 5856 spores wasconducted. Tea and coffee along with the Bacillus coagulans MTCC 5856spores were brewed and then subjected to gastric stress (acid and bile)followed by anaerobic incubation at 37° C. with bile salts to mimichuman conditions. This set of experiment was conducted to understand thespore germination when Bacillus coagulans MTCC 5856 is brewed anddigested by human. Additionally, along with tea and coffee B. coagulansMTCC 5856 was brewed with water soluble prebiotic fibres(fructooligosaccharide, galacto-oligosacharides, inulin andpolydextrose). Results of the experiment revealed that B. coagulans MTCC5856 spores could germinate after brewing and surviving in harshcondition of gastric stress by using tea or coffee as sole nutritionalsource (Table 6). Additionally, there was further increase in thegermination by the addition of water soluble prebiotic fibres (Table 6).This suggested that soluble prebiotic fibres have synergistic effectalong with tea and coffee on the germination of B. coagulans MTCC 5856spores.

TABLE 6 Germination of Bacillus coagulans MTCC 5856 in gastric stressusing tea and coffee or in combination with water soluble prebioticfibres as nutritional source OD at 600 nm % (1:5 dilution) Increase S.No. Probiotic Composition 0 h 24 h in growth 1 Black Tea + B. coagulansMTCC 5856 0.083 ± 0.001 0.156 ± 0.01 46.79 2 Black Tea + FOS + B.coagulans MTCC 5856 0.091 ± 0.002 0.185 ± 0.01 50.81 3 Black Tea + GOS +B. coagulans MTCC 5856 0.085 ± 0.001 0.187 ± 0.02 54.54 4 Black Tea +Inulin + B. coagulans. MTCC 5856 0.088 ± 0.001 0.191 ± 0.01 53.92 5Black Tea + Polydextrose + B. coagulans MTCC 5856 0.092 ± 0.003 0.188 ±0.02 51.06 6 Green Tea + B. coagulans MTCC 5856 0.075 ± 0.001 0.112 ±0.02 33.03 7 Green Tea + FOS + B. coagulans MTCC 5856 0.078 ± 0.0020.125 ± 0.01 37.60 8 Green Tea + GOS + B. coagulans MTCC 5856 0.075 ±0.001 0.145 ± 0.03 48.27 9 Green Tea + Inulin + B. coagulans MTCC 58560.079 ± 0.003 0.141 ± 0.01 43.97 10 Green Tea + Polydextrose + B.coagulans MTCC 5856 0.074 ± 0.002 0.142 ± 0.02 47.88 11 Oolong Tea + B.coagulans MTCC 5856 0.084 ± 0.001 0.123 ± 0.01 31.70 12 Oolong Tea +FOS + B. coagulans MTCC 5856 0.082 ± 0.001 0.162 ± 0.02 49.38 13 OolongTea + GOS + B. coagulans MTCC 5856 0.085 ± 0.003 0.159 ± 0.01 46.54 14Oolong Tea + Inulin + B. coagulans MTCC 5856 0.086 ± 0.001 0.159 ± 0.0145.91 15 Oolong Tea + Polydextrose + B. coagulans MTCC 5856 0.085 ±0.002 0.157 ± 0.01 45.85 16 Coffee + B. coagulans MTCC 5856 0.142 ±0.001 0.361 ± 0.01 60.66 17 Coffee + FOS + B. coagulans MTCC 5856 0.139± 0.001 0.521 ± 0.02 73.32 18 Coffee + GOS + B. coagulans MTCC 58560.154 ± 0.002 0.531 ± 0.03 70.99 19 Coffee + Inulin + B. coagulans MTCC5856 0.145 ± 0.001 0.516 ± 0.01 71.89 20 Coffee + Polydextrose + B.coagulans MTCC 5856 0.144 ± 0.003 0.532 ± 0.02 72.93

Example 3: Beverage Compositions with Bacillus coagulans MTCC 5856

The viability of Bacillus coagulans MTCC 5856 was also tested in otherbeverages like aerated soda/cola, fruit juices, mineral water,chocolate, cocoa, fruit drinks, smoothies, milk shakes, cheese spread,honey, tomato ketchup, jam, mayonnaise, protein drink and ice cream. Theresults are tabulated in tables 7 & 8.

TABLE 7 Stability and recovery of Bacillus coagulans Spores in beveragesSoda drink with Timms Up with Orange Juice with Sprite with MineralWater with B. coagulans B. coagulans B. coagulans B. coagulans B.coagulans MTCC 5856 MTCC 5856 MTCC 5856 MTCC 5856 MTCC 5856 Log₁₀ Log₁₀Log₁₀ Log₁₀ Log₁₀ CFU/ Viability CFU/ Viability CFU/ Viability CFU/Viability CFU/ Viability Time serving (%) serving (%) serving (%)serving (%) serving (%) Expected 9.301 100 9.301 100 9.301 100 9.301 1009.301 100 Recovery After 24 h 8.612 92.59 8.454 92.59 9.217 99.1 8.20488.21 9.26 99.56

TABLE 8 Stability of B. coogulans MTCC 5856 spores during the process ofpreparation of various foods and beverage mix and its recovery S. No.Sample Log₁₀ CFU/serving % Recovery 1 Dry Fruit (Cashew) with B.coagulans MTCC 5856 (20 g serving) 8.781 94.41 2 Chocolate with B.coagulans MTCC 5856 (50 g serving) 9.25 99.45 3 Cheese Spread with B.coagulans MTCC 5856 (50 g serving) 9.26 99.56 4 Honey with B. coagulansMTCC 5856 (50 g serving) 9.13 98.16 5 Mixed Fruit Jam with B. coagulansMTCC 5856 (50 g serving) 8.989 96.65 6 Tomato Ketchup with B. coagulansMTCC 5856 (50 g serving) 9.065 97.46 7 Mayonnaise with B. coagulans MTCC5856 (50 g serving) 8.958 96.31 8 Protein Drink Mix with B. coagulansMTCC 5856 (50 g serving) 9.274 99.71 9 Soy Milk with B. coagulans MTCC5856 (50 g serving) 8.238 88.57 10 Shrikhand with B. coagulans MTCC 5856(50 g serving) 9.064 97.45 11 Ice-cream with B. coagulans MTCC 5856 (30g serving) 7.99 85.90

The results indicated that B. coagulans MTCC 5856 is stable in allbeverage compositions with increased viability and recovery.

Other modifications and variations to the invention will be apparent tothose skilled in the art from the foregoing disclosure and teachings.Thus, while only certain embodiments of the invention have beenspecifically described herein, it will be apparent that numerousmodifications may be made thereto without departing from the spirit andscope of the invention.

We claim:
 1. A beverage composition comprising Bacillus coagulans MTCC5856 in the form of spore and bacterium wherein the bacterial spore hasthe ability to survive extreme stress, temperature and aerationtreatments of the beverage composition, wherein the beverage compositionis tea, wherein the tea is selected from the group consisting of greentea, black tea, oolong tea, yellow tea, white tea, decaffeinated tea,rosehip tea, chamomile tea, jiaogulan tea, peppermint tea, rooibos tea,ginger tea, ginseng tea, or lemon grass tea.
 2. The composition as inclaim 1, wherein the spores comprise viable or heat killed or deadspores of Bacillus coagulans MTCC
 5856. 3. The composition as in claim1, wherein the bacterium comprises viable or heat killed or dead orlysed vegetative cells of Bacillus coagulans MTCC
 5856. 4. Thecomposition as in claim 1, wherein the bacterial spore is heat resistanthaving more than 95% viability after brewing the tea.
 5. The compositionas in claim 4, wherein the spores comprise viable or heat killed or deadspores of Bacillus coagulans MTCC
 5856. 6. The composition as in claim4, wherein the bacterium comprises viable or heat killed or dead orlysed vegetative cells of Bacillus coagulans MTCC 5856.